WO2021018321A2

WO2021018321A2 - Biopesticide and biofertiliser composition

Info

Publication number: WO2021018321A2
Application number: PCT/CU2020/050004
Authority: WO
Inventors: Irene María ALVAREZ LUGO; Ileana Sanchez Ortiz; Idania WONG PADILLA; Danalay SOMONTES SANCHEZ; Rolando MORAN VALDIVIA; Liszoe GALDÓS BETARTEZ; Laritza Caridad DOMINGUEZ RABILERO; Aylin NORDELO VALDIVIA
Original assignee: Centro De Ingenieria Genetica Y Biotecnologia
Priority date: 2019-07-31
Filing date: 2020-07-30
Publication date: 2021-02-04
Also published as: AR119504A1; WO2021018321A3; BR112022001668A2; CU20190071A7; MX2022001282A; CN114501999A; US20220272986A1; EP4005387A2

Abstract

A biopesticide and biofertiliser composition which comprises Pseudoxanthomonas indica, or metabolites of said bacterium, and excipients or diluents. Use of Pseudoxanthomonas indica, or metabolites of said bacterium, for manufacturing a biopesticide or biofertiliser composition. A method for controlling phytopathogens and zoonematodes which comprises the application of an effective amount of Pseudoxanthomonas indica, or its metabolites, to the plant or animal that needs it. A method for promoting plant growth which comprises the application of an effective amount of Pseudoxanthomonas indica, or metabolites of said bacterium, to the soil or substrate, plant or seed.

Description

BIOPESTICIDE AND BIOFERTILIZING COMPOSITION

Technical field

The present invention is related to the field of agriculture, and is based on the application of Pseudoxanthomonas indica as a beneficial microorganism for it. As shown in the invention, the bacteria of this microbial species possess nematicidal, fungicidal, inducer of defensive response, and growth promoting activity in plants, and exert these effects without affecting the environment.

Prior state of the art

Plants are associated with microorganisms that exert biofertilizing and biocontrol action. These microorganisms, with their biofertilizing effect, favor the good condition of the plant through the increase in the availability of nutrients, nitrogen fixation and increase symbiosis. Biocontrol manifests itself with the increase in plant growth, due to the control of pathogenic organisms. This effect develops through mechanisms such as the production of antibiotics, siderophores, antagonist substances of bacteria and fungi, extracellular enzymes and the increase in resistance to biotic and abiotic stress. In general, within the bacterial genera of this type are Pseudomonas, Serratia, Bacillus, Enterobacter and Xanthomonas, and within the fungi Trichoderma can be mentioned (Mabood, Zhou and Smith 2014, Can. J. Plant Sci. 94: 1051-1063).

In particular, one of the most common phytopathogens are nematodes, as they constitute one of the most important pests in agriculture in tropical, subtropical and temperate zones. Among the nematode species, Meloidogyne ssp. it causes crop losses estimated between 1 1 and 25%, in many tropical areas. Annual losses from this concept are estimated at around $ 87 trillion worldwide. Due to their speed and efficiency, chemical nematicides have been the most used method for the control of nematodes. But fumigants, such as methyl bromide, have been banned in many countries, due to their high toxicity to mammals, their ozone-depleting action, and their residual effect. Taking into account these premises, the use of beneficial microorganisms antagonists of phytonematodes is currently being promoted to obtain biological products (Jones et al. 2013, Mol Plañí Pathol 14 (9), 946-961). Among the natural enemies of nematodes are the fungi Paecilomyces lilacinus, Verticillium chlamydosporium, Arthrobotrys oligospora and Trichoderma harzianum. Bacteria such as Pasteuria penetrans, Agrobacterium radiobacter, Bacillus sphaericus, Bacillus subtilis, Bacillus thuringiensis (Dong and Zhang 2006, Plant and Soil 288 (1): 31-45) and Tsukamurella paurometabola strain C924 (Patent EP 0774906 B1) are also found. More recently, strains of the genera Chromobacterium, Burkholderia, and Flavobacterium were patented to modulate nematode infestation.

However, until now there are no fully effective bioproducts, so there is a need to have beneficial microorganisms, towards which resistance is not developed by plant pathogens, and which have a minimal environmental effect.

Explanation of the invention

The present invention solves the aforementioned problem, by providing a biopesticidal or biofertilizing composition comprising Pseudoxanthomonas indica, or metabolites of said bacterial species, and excipients or diluents. The invention demonstrated for the first time that this species can be used as a broad spectrum nematicide and fungicide, and that it is capable of controlling the populations of nematodes and phytopathogenic fungi in the main agricultural crops. The experiments of the invention showed that the biopesticidal composition comprising Pseudoxanthomonas indica reduces the hatching of Meloidoavne eggs "in vitro", compared to that observed in eggs not treated with said bacteria, and that it increases the mortality of nematode larvae. The application to the soil of this bacterium reduces the infestation index by Meloidogyne spp. in tomato, and reduces the number of galls on the roots with a high technical efficiency (ET).

In the context of this invention, a biopesticide is understood to be a product that is used to control agricultural pests, by means of specific biological effects. This differentiates them from chemical pesticides, which have a broader action. As is known to those skilled in this field of the art, biopesticide products comprise a biological control agent, that is, an organism, its genes or its metabolites, for the control of pests (Lacey and Sporleder, 2013, Chapter 16 Biopesticides, Insect Pests of Potato, 463-497pp). In composition Biopesticide of the invention, the biological control effect is exerted by a bacterium or the compounds that it produces.

In the context of this invention, a biofertilizer is understood as a product that contains a beneficial organism, which favors the growth and development of plants, as an alternative to chemical fertilizers, without adverse effects on the ecosystem. As is known to those skilled in the art, biofertilizer products facilitate the availability and absorption of nutrients, as well as the production of plant hormones. In addition, they give the plant tolerance to stress, and increase resistance to pathogens, which results in an improvement of the crop (Bhardwaj et al., 2014 Microbial Cell Phaetones 13:66).

According to the experimental evidences shown, the invention provides an inducer of the defensive response and a promoter of the growth of the treated plants. This indicates Pseudoxanthomonas comprising the microorganism, which is a positive or negative, motile, catalase and oxidase Gram and forms yellow colonies at 24 hours of growth at 28 ^Q C in Tryptone Soya Agar. In the invention, the bacterium was obtained from the rhizosphere of a healthy tomato plant, from a grow house infested with Meloidogyne spp., And it was identified as Pseudoxanthomonas indica by conventional biochemical techniques, and molecular biology. This bacterial species is not new, it has been isolated from soils in other regions of the world. The representative strain of this species, named P15, was isolated from the soil of an open air hexachlorocyclohexane dump in India, and was reported in 201 1 (Kumari et al. 2011, Int J Syst Evol Microbiol 61: 2107-2111 ). The species is commercially available in known collections of microorganisms.

In one embodiment of the invention, the biopesticidal or biofertilizer composition comprises the live or inactivated Pseudoxanthomonas indica bacteria. In the present invention, it is demonstrated that both the culture supernatant, as well as the living bacteria, and their volatile or soluble compounds (metabolites), have nematicidal and / or fungicidal activity.

In the context of the invention, metabolite is understood to be that substance or compound produced by Pseudoxanthomonas that indicates that it is capable of controlling pests in plants and zoonematodes, and / or possesses plant growth stimulating activity. In one embodiment of the invention, the metabolites originally produced by Pseudoxanthomonas indica are obtained via natural, recombinant or by chemical synthesis. In a particular embodiment, the metabolites are volatile or soluble compounds.

The metabolites released by Pseudoxanthomonas indica to the culture medium, and the volatile compounds it produces, have "in vitrd" activity on phytopathogenic fungi: Rhizoctonia solani, Sarocladium oryzae, Stemphylium solani, Phytophthora nicotianae, Bipolaris oryzae and Fusarium oxysporum. In addition, they have plant growth stimulating activity, and increase the length and weight of the plants, the length of the main root and the number of lateral roots and root hairs.

In one embodiment of the invention, the composition further comprises stabilizing, enhancing, eliciting compounds of nematicidal activity or seed coatings. In one embodiment of the invention, the concentration of the bacteria is between 1 x 10 ⁸ and 1 x ^{10 10} colony forming units (ufe) per ml of composition. The biopesticidal composition is useful for the direct or indirect control of phytopathogens and zoonematodes.

In another aspect, the invention comprises the use of Pseudoxanthomonas indica, or metabolites of that bacterium, for the manufacture of a biopesticidal or biofertilizer composition. In one embodiment of the invention, for said use, the concentration of the bacteria is between 1 x 10 ⁸ and 1 x ^{10 10} ufe per ml of composition. In one embodiment of the invention, the biofertilizer composition is formulated as a prepackaged soil, a powder, a liquid, a suspension, a granulate, a nebulizer or an encapsulation.

On the other hand, the invention reveals a method for the control of phytopathogens and zoonematodes that comprises the application of an effective amount of Pseudoxanthomonas indica, or metabolites thereof, to the plant or animal that needs it. In one embodiment of the invention, in said method the bacterium is applied as a suspension or a concentrate of the live or inactivated strain. In a particular embodiment, in said method, the bacterium is formulated as a prepackaged soil, a powder, a liquid, a suspension, a granulate, a nebulizer or an encapsulation. In one embodiment, in the method of the invention, the strain is useful to reduce the degree of infestation produced by nematodes or phytopathogenic fungi in plants.

Another object of the invention is a method for the stimulation of plant growth that comprises the application of an effective amount of Pseudoxanthomonas indica, or metabolites thereof, to the soil, substrate, plant, or seed. In said method, optionally, the bacterium is formulated as a prepackaged soil, a powder, a liquid, a suspension, a granulate, a nebulizer or an encapsulation.

Brief description of the figures

Figure 1. Phylogenetic tree of strain H32, according to the Neighbor-joining method based on the sequence of genes that encode ribosomal ribonucleic acid (rRNA) 16s, showing the relationship of strain H32 with representative members of the Pseudoxanthomonas family. As microorganisms outside the group, Luteimonas mephitis B1953 / 27, Arenimonas donghaensis H03-R19, Lysobacter enzymogenes DSM 2043T and Thermomonas haemolytica A50-7-3 were used. Resampling values greater than 70% appear at branch nodes (these are expressed in% of 1 000 replicates). The bar represents two substitutions for every 100 nucleotides.

Figure 2. Effect of volatile organic compounds (abbreviated VOCs, volatile organic compounds) on the growth of the fungus Bipolaris oryzae. H32, H36 and H42: Bacterial strains under study. Control: Untreated group. Different letters indicate statistically significant differences, according to Duncan's test, p <0.05.

Figure 3. Length of the branches (A) and weight of the plants (B) of Arabidopsis thaliana, after 14 days of exposure to VOCs of Pseudoxanthomonas indica strain H32 (H32) or of the culture medium Tryptone Soy Broth (Control) . The asterisk indicates statistically significant differences, according to the Student's test p <0.05.

Figure 4. Length of the main root in Arabidopsis thaliana plants, after eight days of co-cultivation with Pseudoxanthomonas indica, strains H32, H36 and H42, or with MgSO ₄ (10 mmol / L) as negative control (Control) . Different letters indicate statistically significant differences, according to Duncan's test, p <0.05. Figure 5. Number of lateral roots in Arabidopsis thaliana plants exposed to Pseudoxanthomonas indica, strains H32, H36 and H42, or to MgSO ₄ (10 mmol / L) as negative control (Control). Different letters indicate statistically significant differences, according to Duncan's test, p <0.05.

Figure 6. Number of root hairs in 1 cm of the main root of Arabidopsis thaliana plants exposed to Pseudoxanthomonas indica, strains H32, H36 and H42, or to MgSO ₄ (10 mmol / L) as a negative control (Control). Different letters indicate statistically significant differences, according to Duncan's test, p <0.05.

Figure 7. Effect of Pseudoxanthomonas indica, strain H32, applied to roots, on the expression of defense genes PR1 and PDF1.2 in Arabidopsis thaliana leaves. Experimental groups: Control plants treated with water (C) and plants treated with the bacterial strain H32 (H32). Different letters indicate statistically significant differences, according to Duncan's test, p <0.05.

Figure 8. Effect of Pseudoxanthomonas indica, strain H32, applied to roots, on the expression of defense genes: osmotin (panel A), hydroperoxide lyase (HPL) (panel B), chitinase (panel C), glutathione peroxidase (GPx) (panel D), PR-1 (panel E), alloy oxide synthase (AOS) (panel F) and phenylalanine-ammonium lyase (FAL) (panel G). Different letters indicate statistically significant differences, according to Duncan's test, p <0.05. The expression of the genes was measured at the systemic level, in tomato leaves.

Detailed exposition of embodiments / Exemplary embodiments

Example 1. Isolation and conservation of bacterial strains with nematicidal properties.

For the isolation of bacterial strains with nematicidal properties, rhizospheric soil samples were collected from less affected tomato plants, in a grow house with high infestation by Meloidogyne spp. in Ciego de Ávila. First, a selective pre-enrichment was carried out. For this, one gram of rhizosphere soil was taken and incubated for 48 hours at 30 ^Q C in M9 minimal medium containing 100 Meloidogyne eggs per ml of culture, as sole source of carbon and energy. From this preparation, serial dilutions were made which were inoculated in Petri dishes, with M9 minimal medium with 0.05% chitin; for the selection of strains. The colonies with halo, which showed different morphological characteristics, were selected and named with an H followed by the consecutive number of the isolate. Next, a second selection of the colonies was carried out, by determining the desulfurase and gelatinase activity. The desulfurase activity is observed by blackening of a filter paper soaked in lead acetate, placed on the colonies in the Petri dish with Tryptone Soy Agar, due to the fact that the colonies produce hydrogen sulfide. The Gelatinase activity is evaluated by observing hydrolysis halos, after 24 hours of growth on nutrient agar with 0.5% gelatin, and the addition of Frazier's reagent.

Strains H32, H36 and H42 after 24 hours incubation at 30 ^Q C in Tryptone Soya Agar, colonies showed 3 to 5 mm, opaque light yellow, circular in shape and convex elevation. The edges of these colonies were whole, had a smooth and shiny surface, and their consistency was creamy. For the conservation of the isolates, a colony was grown for 12 hours in Tryptone Soy Broth, glycerol was added as a cryoprotectant (at 20% final concentration (v / v)), and these samples were frozen at -70 ^° C.

Example 2. Taxonomic identification of the isolated bacteria.

For identification, the Gram stain, the oxidase and catalase test, and the Hugh Leifson test were performed, according to the Bergey Manual of Systematic Bacteriology (Vos et al. 2009, Springer Dordrecht Heidelberg London New York, 2nd Edition, Vol 3, 1450 pp ISBN: 978-0-387-95041 -). Strains H32, H36, and H42 were determined to be Gram negative, motile, aerobic, and oxidatively metabolized bacilli. Conventional tests, carried out using the API 20NE kit (Biomeriux), showed a profile that did not coincide with any of those in the kit's identification catalog. The results of the biochemical tests are shown in Table 1.

Table 1. Results of the biochemical tests carried out using the API 20NE. Test Result

Nitrate reduction to Nitrite +

Indole production

Production of glucose acid

Arginine dihydrolase

Urease

Esculin hydrolysis +

Gelatin hydrolysis +

b-galactosidase +

Assimilation of:

D-Glucose +

L-Arabinose D-Mannose +

D-Mannitol

N-acetyl glucosamine +

D-Maltose +

Gentibiosa

Caproic acid

Adipate

Malate +

Citrate

Phenyl acetate acid

Oxidase +

The strains referred to in Example 1 were also characterized by molecular biology, by sequencing a fragment of the gene that codes for 16S rRNA. The oligonucleotides that were used as primers for the Polymerase Chain Reaction were 63F and 1387R (Marchesi et al. 1998 Applied and Environmental Microbiology 64 (2): 795-9). The sequences were compared with those available in the GenBank database, from the National Center for Biotechnology Information in the United States, using the bioinformatics package MEGA 7.0 (Molecular Evolutionary Genetics Analysis Software Version 7.0, Center for Evolutionary Functional Genomies, The Biodesign Institute, Arizona State University). The phylogenetic tree represented in Figure 1, which was obtained by the Neighbor-joining method, confirmed that the strains H32, H36 and H42 formed a coherent group with the members of the genus Pseudoxanthomonas of the phylum Bacteroidetes, and an intragenus branch with Pseudoxanthomonas indica . Luteimonas mephitis B 1953/27, Arenimonas donghaensis FI03-R19, Lysobacter enzymogenes DSM 2043T and Thermomonas haemolytica A50-7-3 were used as out-of-group microorganisms. Resampling values greater than 70% appear at branch nodes. Figure 1 shows only what is related to the H32 strain. The phylogenetic tree obtained by the maximum probability method had a similar topology as that of Neighbor-joining.

The analysis of the distance matrix allowed to determine the greater proximity of the 16S rRNA gene sequence of the strains under study with Pseudoxanthomonas. indicates P15, EF424397.1. According to the results of the phylogenetic analysis, it is inferred that the studied strains belong to the Pseudoxanthomonas indica species, and the results of their biochemical tests correspond to the main characteristics described for this bacterial species.

Example 3. Nematicidal activity "in vitro" of the bacteria corresponding to the selected strains.

In vitro tests were carried out with eggs and juveniles of the phytonmatode Meloidogyne spp., And the nematicidal activity of live Pseudoxanthomonas indica cells was determined. For this, the egg masses were extracted from the roots of tomato plants affected by this plague, the masses were disaggregated with 0.5% sodium hypochlorite; and three replicas (of 100 eggs) were placed for each sample to be tested. To obtain the larvae, the eggs were incubated at a temperature of 28-30 ° C, in sterile deionized water, for 6 days. Control samples were also prepared, with 100 eggs and 100 larvae of this nematode contained only in 1 ml of sterile peptone water (1% (w / v)). As a criterion of activity against Meloidogyne spp. Eggs, the percentage of inhibition of the hatching of the eggs was determined. As a criterion of activity against larvae or juveniles of this nematode, the percentage reduction in their survival was calculated.

Pseudoxanthomonas indica whole live cell samples were prepared from a fresh culture of each strain, at which the bacterial concentration was determined as ufe per ml. 1 ml of the culture was taken, in duplicate, the cells were separated by centrifugation, and washed with peptone (1%) in water. Subsequently, three serial dilutions, 1: 100, were made in the same solution, which were used in the tests. Tables 2 and 3 show the effect of two of the dilutions of Pseudoxanthomonas indica on the eggs and larvae of nematodes.

Table 2. Percent inhibition of egg hatching of the nematode Meloidogyne spp. at two concentrations of viable cells of Pseudoxanthomonas indica.

Strain Hatch inhibition

5x10 ^b cfu / ml 5x10 ⁴ cfu / ml

H32 83% 89% H36 75% 80%

H42 74% 85%

Witness 20% 22%

Table 3. Percentage reduction in the survival of larvae of the nematode Meloidogyne spp. at two concentrations of live cells of Pseudoxanthomonas indica.

Strain Reduced survival of larvae

5x10 ⁶ cfu / ml 5x10 ⁴ cfu / ml

H32 50% 55%

H36 43% 45%

H42 38% 40%

Control 15% 20%

Therefore, it is evidenced that Pseudoxanthomonas indica inhibits the hatching of Meloidogyne spp. It also reduces the survival of the larvae of this nematode.

Example 4. Carbohydrate utilization profiles and enzymatic activity of Pseudoxanthomonas indica.

To determine the carbohydrates used by the strain H32 of Pseudoxanthomonas indica, as the sole source of carbon and energy, the CH50 kit from Biomeriux was used. Bacteria of the Pseudoxanthomonas indica strain H32 oxidized the following carbohydrates: D-Glucose, D-Fructose, D-Mamnosa, N-acetyl-Glucosamine, D-Maltose, D-Lactose, D-Sucrose, as reported for type strain of this species (Pseudoxanthomonas indica P15).

For the determination of the enzymatic profile of said strain of Pseudoxanthomonas indica, the production of chitinases was detected, by means of growth in M9 medium with colloidal chitin at 0.5%. Protease production was detected by plating with Nutrient Agar and 0.5% gelatin, and subsequent development with Frazier's reagent. In addition, the APIZYM ™ kit was used for the detection of phosphatases, esterases, lipases, glucuronidases, galactosidases, fucosidase, mannosidase, arylases, N-acetyl-p-glucosaminidase and Naphthol-AS-BI-phosphohydrolase. The results of these determinations are shown in Table 4, and they demonstrate that the bacteria of the Pseudoxanthomonas indica strain H32 possess an oxidative metabolism, and that they are capable of degrading a large number of organic compounds and other complex chemical compounds.

Table 4. Enzymes released by Pseudoxanthomonas indica strain H32.

Enzyme Result

Chitinases +

Proteases +

Esterase (C4) +

Lipase esterase (C8) +

Lipase (C14)

Trypsin +

to chymotrypsin +

b-glucosidase

N-acetyl ^ -glucosaminidase +

Alkaline phosphatase +

Leucine arylamidase +

Valine arylamidase +

Cystine arylamidase +

Acid phosphatase +

Naphthol-AS-BI-phosphohydrolase +

+: presence

absence

Example 5. Effect of Pseudoxanthomonas indica on infestation by Meloidogyne spp. in Solanum lycopersicum.

A test was carried out to evaluate the nematicidal activity "in vivo" of Pseudoxanthomonas indica in tomato plants (Solanum lycopersicum). They were transplanted into pots with 1 kg of substrate, and infested with 500 eggs of Meloidogyne spp. The bacteria were grown in Tryptone Soy Broth medium for 24 h, and serial dilutions were made in deionized water, to apply it to the plants at concentrations between 4x10 ⁷ and 2x10 ⁴ cfu / g of substrate. The scheme that was used was of two applications. An application was made 3 days after infestation of the substrate with nematodes, and 7 days later the tomato seedlings were transplanted. The second application was made at 14 days after transplant. As a positive control, the bacterial strain C-924 was used, which has known properties as a nematicide (European Patent EP 0774906 B1). In this experiment, deionized water was used as a control (negative control).

At 35 days after transplantation, the tomato plants were extracted and the degree of infestation of the roots was determined, using the Bridge and Page scale, from 0 to 1 degrees. From these results, the ET (Wong et al. (2017) Biotecnología Aplicada, 34: 4301-43) of each concentration of strain H32 was determined. The bacterium was effective against nematodes of the genus Meloidogyne, in all the treatments that were evaluated, as shown in Table 5.

Table 5. Degrees of infestation of the roots of Solanum lycopersicum and ET of the treatments with Pseudoxanthomonas indica.

Treatment cfu / g ET Grading * (%)

TTÓ 4x10 ^/ 1 .38 75.32

T 1 1 4x10 ⁶ 1, 89 66.10

T 12 4x10 ⁵ 1, 29 76.92

T 13 4x10 ⁴ 2.38 57.37

T 14 2x10 ⁴ 2.43 56.41

Strain C-924 2x10 ⁸ 3.00 46.15

WITNESS 5.60 0.0

^* ET: Technical Efficiency

Therefore, bacteria of the species Pseudoxanthomonas indica reduce the infestation index by Meloidogyne spp. with an ET greater than 50%.

Example 6. Antifungal activity of the metabolites released to the culture medium of Pseudoxanthomonas indica.

To determine the antifungal activity of the metabolites released into the culture medium of the Pseudoxanthomonas indica strain H32, a disc, approximately 7 mm, of culture medium containing the fungus was first placed in the center of a Petri dish with medium. Potato Dextrose Agar (PDA). On both sides, 10 pL of a bacterial suspension of strain H32 in Tryptone Soya Broth was deposited in the form of a line. In the control treatment, only the same culture medium was placed. 2 replications were made in each case. At 5 days of incubation at 28 ^Q C, the diameter of the colonies of the fungi was measured and the percentage of relative growth with respect to the control was determined as:

% growth inhibition = ((diameter of the control colony - diameter of the treated colony) / (diameter of the control colony -7)) ^* 100.

The results are shown in Table 6, which indicate that Pseudoxanthomonas indica stopped the development of a group of phytopathogenic fungi that affect crops of great economic importance.

Table 6. Inhibition of the growth of phytopathogenic fungi by the metabolites released by Pseudoxanthomonas indica.

Growth inhibition fungus (%)

Phyizoctonia solani 77.9

Sarocladium oryzae 47.8

Stemphylium solani 72.0

Phytophthora nicotianae 92.6

Bipolaris oryzae 80.9

Fusarium oxysporum cubes 43.9

Example 7. Antifungal activity of volatile organic compounds produced by Pseudoxanthomonas indica.

A system was set up with two Petri dish bottoms. In the lower part, with PDA medium, a disk, approximately 7 mm, of the Bipolaris oryzae fungus was placed. In the upper part, with Tryptone Soy Agar, 100 ml_ of a 12 hour culture of the bacterial strains H32, H36 and H42 were dispersed in Tryptone Soy Broth. In the control treatment, only 100 ml of Tryptone Soy Broth were dispersed. 2 replications were made in each case. The system was sealed with a Parafilm ™ film, and incubated at 28 ^° C. At 5 days, the diameter of the fungal colonies was measured. Growth inhibition relative to control treatment was determined as:

% Growth inhibition = ((diameter of the control colony - diameter of the treated colony) / (diameter of the control colony -7)) ^* 100

The three strains studied inhibited the growth of Bipolaris oryzae by more than 50%, without physical contact with the pathogen, as shown in Figure 2. Therefore, the volatile compounds of Pseudoxanthomonas indica have antifungal activity.

Example 8. Production of volatile plant growth promoting compounds by the bacterium Pseudoxanthomonas indica.

To demonstrate growth-promoting activity in plants, Arabidopsis thaliana seeds (approximately 100 seeds per treatment) were sterilized with a mixture consisting of 1 ml_ of sodium hypochlorite (2% (v / v)) and 3 ml_ of newt X100 ( 1% (v / v)), to then arrange for 8 minutes at 37 ^Q C, with stirring. Then they washed about 5 times with sterile distilled water, and stored at 4 ^Q C for 4 days. They were placed in 150 mm diameter Petri dishes, with MS medium, to which a small Petri dish (50 mm diameter) with Tryptone Soy Agar was placed towards the center. After two days of plant germination, the medium Tryptone soy agar was inoculated with 10 pi _¬ a culture of the microorganism under study, previously grown for 12 h in tryptone soy broth at 37 ^Q C. The system was sealed with Parafilm ™ film, and incubated at 20 ± 2 ^Q C with a photoperiod of 16 h light and 8 h dark.

Samples were taken after 14 days to evaluate the length and weight of the branches and the number of leaves. Arabidopsis thaliana plants, after 14 days of incubation in Petri dishes containing Pseudoxanthomonas indica, showed better growth than the control without bacteria (Figure 3). This evidenced the stimulation of the growth of said plants by the volatile compounds produced by said bacteria.

Example 9. Effect of soluble compounds released by Pseudoxanthomonas indica on the morphology of the roots.

Arabidopsis thaliana var Col 0 seeds were exposed to soluble compounds released to the culture medium by Pseudoxanthomonas indica. Previously, the seeds were sterilized by immersion for 8 minutes at 37 ^Q C, with stirring, in a solution containing 1 mL of sodium hypochlorite (2% (v / v)) and 3 pL of Triton X100 (1% ( v / v)). The seeds were washed five times with sterile distilled water, and kept at 4 ^Q C for 4 days.

10 seeds were deposited, in a straight line and equidistant, in the upper end of a 100 mm Petri dish, containing MS medium. Plates were placed vertically in the lighted room at a temperature of 22 ± 2 ^Q C and a photoperiod of 16 h light and 8 h dark. 4 days after germination of the 240 ml_ of a bacterial suspension of the H32, H36 and H42 strains were added to the culture medium, at a distance of 2-3 cm from the lower end of the roots. These suspensions were prepared from a culture medium in Tryptone Soya Broth grown at 28 ^Q C for 18 h, which was centrifuged and resuspended in MGSC> April 10 mmol / L. The system was sealed with Parafilm ™, and incubated in the lighted room, under the same conditions. As a negative control, the MgSO ₄ 10 mmol / L solution was used. 3 replicas were mounted in each case.

At 8 days of co-cultivation, the length of the primary root, the number of lateral roots, and the number of root hairs in 1 cm of primary root, from the lower end upwards, were evaluated. For this evaluation, the stereoscope and the inverted microscope were used. The roots of the plants co-cultivated with the bacterial strains presented different morphology, compared to the control group. This showed the possible alterations in the architecture of the roots, induced by this bacterium. The length of the primary root, in the plants subjected to the treatments with the bacteria, was greater (with statistical significance p <0.05) than the length of the same in the control treatment (Figure 4).

The number of lateral roots and the number of root hairs were also significantly higher than in the control treatment {p <0.05), as seen in Figure 5 and Figure 6. Therefore, the application of Pseudoxanthomonas indicates increases the surface area of the roots and, consequently, the possibility of absorption of nutrients.

Example 10. Evaluation of the ability of Pseudoxanthomonas indica to induce the expression of immune response markers in Arabidopsis thaliana and tomato.

The seeds of A. thaliana were incubated in water at 4 ° C for four days, and then sown in a substrate composed of sand: peat (1: 1). Six weeks after germination, they were transplanted into small pots containing the same substrate composition. A week later, the pots were randomly divided into two groups. The first group was treated with bacteria of the H32 strain, at a rate of 1 mL per plant, with a suspension containing 10 ⁹ cfu / mL, in saline solution. The second group (control) was only treated with 1 mL of saline solution. Four applications were made, at intervals of seven days. After two hours After the last application, samples of leaves and roots of the plants of each treatment were collected, and they were immediately frozen in liquid nitrogen. The expression of the genes PR1 (marker of immune response mediated by salicylic acid) and PDF1.2 (marker of immune response mediated by jasmonic acid) was determined by the quantitative real-time polymerase chain reaction (RT-qPCR). The ubiquitin gene was used as internal control. As shown in Figure 7, an increased expression of the PDF1.2 gene was observed in the leaves of the plants treated with bacteria of the H32 strain, with respect to the control of the experiment. The expression level of the PR1 gene was not different from that observed in the control group. Therefore, bacteria of the species

Pseudoxanthomonas indica induced the expression of PDF1.2, systemically, which is reported as a defense mechanism related to resistance against infestation by nematodes.

Tomato trial

The objective of the study was to evaluate the capacity of Pseudoxanthomonas indica to modulate the immune response in tomato (Solanum lycopersicum), at the systemic level. Bacteria of the H32 strain were applied as a cell suspension of 10 ⁹ cfu / mL, in the rhizosphere of the plants. Samples were taken at 12 h, 3 days and 7 days after applying the bacteria. At each collection time, samples of six plants of each treatment were randomly taken, which were distributed in two replicas, to homogenize the variations. Once the samples were collected, they were immediately conserved in liquid nitrogen, until further processing. Using the RT-qPCR method, the relative expression of the defense genes phenylalanine-ammonium lyase (FAL), glutathione peroxidase (GPx), osmotin, chitinase, hydroperoxide lyase (HPL), alene oxide synthase (AOS) and PR1 was determined. in sheets. Actin was used as an internal test control.

Figure 8 shows that Pseudoxanthomonas indica can induce, in the early stages of interaction with the plant, the expression of the osmotin, HPL, chitinase, GPx and FAL genes. On the other hand, suppression of the PR1 gene was observed at 12 hours and 3 days after applying the bacteria to the plants, and overexpression of the same gene at seven days, with respect to the plants of the control group. The expression of the AOS gene, in the evaluated times, did not increase with respect to the controls. The results obtained show that Pseudoxanthomonas indica can modulate the plant immune system, to promote resistance to attack by pathogens. Example 11. Obtaining a formulation of Pseudoxanthomonas indica and demonstration of its stability.

A formulation was obtained consisting of a concentrate of Pseudoxanthomonas indica, strain H32, as an active ingredient, yeast extract as a source of organic matter rich in amino acids, vitamins and growth factors, a surfactant and water. The final composition of the formulation was: Yeast extract 70.4%; Sucrose 1.4%; DG-158 ™ glanapon at 3.2%, and 20% of dry biomass of the microorganism Pseudoxanthomonas indica. In the composition, the bacteria remain at a concentration between 10 ⁹ and 10 ¹ ° cfu / mL. The residual moisture of the composition was 5.0%. A real-time stability study was carried out on 3 batches of the formulation, for which the viable cell count and the nematicidal activity against the phytonmatode Meloidogyne incognita were determined, in pots, 6 and 12 months after the formulation was stored.

Table 7 shows the number of viable cells and the ET of the formulation for the indicated times. This reflects that the formulation or composition for the biological control of phytonmatodes is stable for at least 12 months.

Table 7. Stability study of the formulation.

ET ^* : Technical efficiency

Example 12. "In vivo" evaluation of the nematicidal effect of Pseudoxanthomonas indica on three species of phytonmatodes.

A procedure was carried out, at the level of the grow house, to evaluate the nematicidal effect "in vivo" of the formulation described in Example 1 1, on the Meloidogyne incognita, Meloidogyne arenaria and Radopholus similis species. The tests with the Meloidogyne incognita and Meloidogyne arenaria species were carried out with tomato plants (Solanum lycopersycum), and the test with Radopholus similis in banana plants (Musa spp.). The tomato and banana seedlings were transplanted into pots with 2 kg of substrate composed of sand: peat (1: 1). Before transplantation, the substrate was inoculated with 3 000 eggs of Meloidogyne incognita, 3 000 eggs of Meloidogyne arenaria or 500 specimens of Radopholus similis. In the trials with Meloidogyne incognita and Meloidogyne arenaria, the scheme used was two applications; the first 3 days after the infestation of the substrate. The second application was made 14 days after transplantation. At 35 days after transplantation, the plants were extracted, and the degree of infestation was determined. For banana plants infested with Radopholus similis, 3 applications were made: at 15, 49 and 63 days. At 80 days after transplantation, the amount of nematodes per 100 g of root was evaluated. In the two cultures, from these results, the ET of the Pseudoxanthomonas indica formulation was determined. As shown in Table 8, the bacterium was effective against the three nematode species evaluated.

Table 8. Technical efficiency of the Pseudoxanthomonas indica formulation against various species of phytonmatodes.

ET ^* : Technical efficiency

Claims

CLAIMS BIOPESTICIDE AND BIOFERTILIZING COMPOSITION

A biopesticidal or biofertilizer composition comprising Pseudoxanthomonas indica, or metabolites of said bacterial species, and excipients or diluents.

The composition of claim 1 comprising the live or inactivated Pseudoxanthomonas indica suspension or concentrate.

3. The composition of claim 1 wherein the metabolites are obtained naturally, recombinantly or by chemical synthesis.

4. The composition of claim 1 wherein the metabolites are volatile or soluble compounds.

The composition of claim 1 which additionally comprises stabilizing compounds, enhancers, elicitors of nematicidal activity or seed coatings.

6. The composition of claim 1 wherein the concentration of Pseudoxanthomonas indica is between 1 x 10 ⁸ and 1 x ^{10 10} cfu / ml.

7. The composition of claim 1 where the biopesticide is used for the direct or indirect control of phytopathogens and zoonematodes.

8. Use of Pseudoxanthomonas indica, or metabolites of said bacterial species, for the manufacture of a biopesticidal or biofertilizing composition.

9. The use of claim 8 wherein the concentration of Pseudoxanthomonas indica is between 1 x 10 ⁸ and 1 x ^{10 10} cfu / ml.

10. The use of claim 8 where the biopesticide is used for the direct or indirect control of phytopathogens and zoonematodes.

1 1. The use of claim 8 wherein the biofertilizer composition is formulated as a prepackaged soil, a powder, a liquid, a suspension, a granulate, a nebulizer or an encapsulation.

12. Method for the control of phytopathogens and zoonematodes that comprises the application of an effective amount of Pseudoxanthomonas indica, or metabolites of said bacterial species, to the plant or animal that needs it.

The method of claim 12 wherein Pseudoxanthomonas indica is applied as a suspension or a concentrate of said live or inactivated bacteria.

The method of claim 12 wherein Pseudoxanthomonas indica is formulated as a prepackaged soil, a powder, a liquid, a suspension, a granulate, a nebulizer or an encapsulation.

15. The method of claim 12 wherein Pseudoxanthomonas indica is used to reduce the degree of infestation produced by nematodes or phytopathogenic fungi in plants.

16. Method for the stimulation of plant growth comprising the application of an effective amount of Pseudoxanthomonas indica, or metabolites of said bacterial species, to the soil, substrate, plant, or seed.

17. The method of claim 16 wherein Pseudoxanthomonas indica is formulated as a prepackaged soil, a powder, a liquid, a suspension, a granulate, a nebulizer or an encapsulation.